Phthalates are ubiquitous environmental contaminants present in soft plastics and PVC-containing devices, resulting in significant human exposure through contamination of food and medical products. The recently completed study by the National Toxicology Program Center for the Evaluation of Risks to Human Reproduction noted there was "serious concern" that some human phthalate exposures are sufficiently high to adversely affect male reproductive function. Despite being the most studied testicular toxicant, the mechanism of phthalate-induced testicular injury is not known. Therefore, the long-term objective of this research is to determine the mechanism of phthalate-induced testicular injury in molecular detail. Hallmarks of phthalate testicular injury are an initial action on Sertoli cells, disruption of Sertoli cell FSH-induced cAMP generation, and the loss of Sertoli-germ cell adhesion. These historical data suggest that both signaling from G protein-coupled receptors and Sertoli-germ cell adhesion proteins are proximal targets of phthalate injury. Recently, putative Sertoli cell G protein-coupled receptors that are cell-cell adhesion proteins (flamingos) were discovered in testis, making such proteins attractive phthalate targets. The specific down-regulation of testicular flamingo mRNA during early stages of phthalate exposure confirmed that flamingo biology was compromised by phthalates. Because of these observations, this grant proposal investigates the mechanism of phthalate-induced testicular injury by examining the following working hypothesis: phthalates induce the phosphorylation, internalization and degradation of flamingo l and 2 protein leading to Sertoli-germ cell detachment and germ cell apoptosis. Three specific aims will test this hypothesis by 1) localizing flamingo 1 and 2 to specific testis cell types, 2) examining both qualitative and quantitative changes in flamingo proteins during phthalate exposure, and 3) determining if disruption of flamingo protein mimics the phenotype produced by phthalate injury.